Peter Crack | University of Melbourne (original) (raw)

Papers by Peter Crack

Background and Purpose-We have previously identified an increased susceptibility of Gpx1 Ϫ/Ϫ mice... more Background and Purpose-We have previously identified an increased susceptibility of Gpx1 Ϫ/Ϫ mice to increased infarct size after middle cerebral artery occlusion (MCAO). This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. Methods-Gpx1 Ϫ/Ϫ mice were exposed to transient MCAO and reperfusion by intraluminal suture blockade. Protein expression of the p65 subunit of transcription factor nuclear factor-B (NF-B) was examined by immunohistochemistry and Western Analysis. NF-B DNA-protein activity was assessed by electrophoretic mobility shift assays (EMSA). Wild-type and Gpx1 Ϫ/Ϫ mice were exposed to MCAO with or without the NF-B inhibitor, pyrrolidinedithiocarbamate (PDTC). Results-Upregulation of the p65 subunit of NF-B and subsequent p65 phosphorylation at serine 536 was detected in the Gpx1 Ϫ/Ϫ brains after stroke. EMSAs revealed that increased ischemia-enhanced DNA binding of NF-B was observed in Gpx1 Ϫ/Ϫ mice compared with wild-type. Supershift assays indicated that the p50 and p65 subunits participated in the bound NF-B complex. The NF-B inhibitor PDTC, a potential antioxidant, was able to afford partial neuroprotection in the Gpx1 Ϫ/Ϫ mice.

Journal of the autonomic nervous system, Jan 8, 1995

Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of n... more Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of nitric oxide synthase (NOS) immunoreactive nerve cells that project to prevertebral ganglia from the gastrointestinal tract and spinal cord of the guinea pig. An antiserum was raised against the neuronal form of NOS by selecting an amino-acid sequence specific to this form as immunogen. The antiserum recognised a single band at 150 kDa on Western blots of rat brain extract. Enteric nerve cells that were labelled by Fast Blue injected into the coeliac ganglion were not NOS immunoreactive in the small intestine, whereas 40-70% were reactive in the large intestine. Retrograde dye injected into the inferior mesenteric ganglion labels cells in the colon and rectum; 60-70% were immunoreactive for NOS. The NOS-immunoreactive nerve fibres arising in the intestine appear to end selectively around somatostatin-immunoreactive nerve cells in the coeliac and inferior mesenteric ganglia. Preganglionic ...

Advanced Healthcare Materials, 2014

Brain repair following disease and injury is very limited due to diffi culties in recruiting and ... more Brain repair following disease and injury is very limited due to diffi culties in recruiting and mobilizing stem cells towards the lesion. More importantly, there is a lack of structural and trophic support to maintain viability of the limited stem/progenitor cells present. This study investigates the effectiveness of an injectable gelatin-based hydrogel in attracting neural progenitor cells (NPCs) from the subventricular zone (SVZ) towards the implant. Glial cell-line-derived neurotrophic factor (GDNF) encapsulated within the hydrogel and porosity within the hydrogel prevents glial scar formation. By directly targeting the hydrogel implant towards the SVZ, neuroblasts can actively migrate towards and along the implant tract. Signifi cantly more doublecortin (DCX)positive neuroblasts surround implants at 7 d post-implantation (dpi) compared with lesion alone controls, an effect that is enhanced when GDNF is incorporated into the hydrogels. Neuroblasts are not observed at the implant boundary at 21 dpi, indicating that neuroblast migration has halted, and neuroblasts have either matured or have not survived. The development of an injectable gelatin-based hydrogel has signifi cant implications for the treatment of some neurodegenerative diseases and brain injuries. The ability of GDNF and porosity to effectively prevent glial scar formation will allow better integration and interaction between the implant and surrounding neural tissue. and 15.5 units L −1 ), and immersed in PBS at 37 °C for 2 d to reach swelling equilibrium. The swollen disks were gently blotted dry on the surface and weighed to measure the wet weight ( M wet ). The disks were then lyophilized and weighed to obtain the dry weight ( M dry ). The swelling ratio (SR) was determined according to Equation , Adv. Healthcare Mater. 2014,

Neuroscience Research, 2009

Journal of Alzheimer's disease : JAD, 2014

The brain transcriptome of Alzheimer's disease (AD) reflects the prevailing disease mechanism... more The brain transcriptome of Alzheimer's disease (AD) reflects the prevailing disease mechanism at the gene expression level. However, thousands of genes have been reported to be dysregulated in AD brains in existing studies, and the consistency or discrepancy among these studies has not been thoroughly examined. Toward this end, we conducted a comprehensive survey of the brain transcriptome datasets for AD and other neurological diseases. We first demonstrated that the frequency of observed dysregulation in AD was highly correlated with the reproducibility of the dysregulation. Based on this observation, we selected 100 genes with the highest frequency of dysregulation to illustrate the core perturbation in AD brains. The dysregulation of these genes was validated in several independent datasets for AD. We further identified 12 genes with strong correlation of gene expression with disease progression. The relevance of these genes to disease progression was also validated in an in...

Redox Rep, 2003

Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidati... more Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidative metabolism and/or exposure to environmental toxins. Antioxidants limit this damage, yet peroxidative events occur when oxidant stress increases. This arises due to increased radical formation or decreased antioxidative defenses. The two-step enzymatic antioxidant pathway limits damage to important biomolecules by neutralising superoxides to water. However, an imbalance in this pathway (increased first-step antioxidants relative to second-step antioxidants) has been proposed as etiological in numerous pathologies. This review presents evidence that a shift in favor of hydrogen peroxide and/or lipid peroxides has pathophysiological consequences. The involvement of antioxidant genes in the regulation of redox status, and ultimately cellular homeostasis, is explored in murine transgenic and knockout models. The investigations of Sod1 transgenic cell-lines and mice, as well as Gpx1 knockout mice (both models favor H(2)O(2) accumulation), are presented. Although in most instances accumulation of H(2)O(2) affects cellular function and leads to exacerbated pathology, this is not always the case. This review highlights those instances where, for example, increased Sod1 levels are beneficial, and indicates a role for superoxide radicals in pathogenesis. Studies of Gpx1 knockout mice (an important second-step antioxidant) lead us to conclude that Gpx1 functions as the primary protection against acute oxidative stress, particularly in neuropathological situations such as stroke and cold-induced head trauma, where high levels of ROS occur during reperfusion or in response to injury. In summary, these studies clearly highlight the importance of limiting ROS-induced cellular damage by maintaining a balanced enzymatic antioxidant pathway.

Journal of Neural Transmission, 2009

Microarray analysis was used to delineate gene expression patterns and profile changes following ... more Microarray analysis was used to delineate gene expression patterns and profile changes following traumatic brain injury (TBI) in mice. A parallel microarray analysis was carried out in mice with TBI that were subsequently treated with minocycline, a drug proposed as a neuroprotectant in other neurological disorders. The aim of this comparison was to identify pathways that may be involved in

Neurochemistry International, 2013

Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (PD) is a complex dis... more Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (PD) is a complex disease, with genetics and environment contributing to the disease onset. Recent studies of causative PD genes have confirmed the involvement of cellular mechanisms engaged in mitochondrial and UPS dysfunction, oxidative stress and apoptosis in the progressive degeneration of the dopaminergic neurons in PD. In addition, clinical, epidemiological and experimental evidence has implicated neuroinflammation in the disease progression. This review will discuss neuroinflammation in PD, with particular focus on the genetic and toxin-based models of the disease. These studies have confirmed elevated oxidative stress and the pro-inflammatory response occurs early in the disease and these processes contribute to and/or exacerbate the nigro-striatal degeneration. In addition, the experimental models discussed here have also provided strong evidence that these pathways are an important link between the familial and sporadic causes of PD. The potential application of anti-inflammatory interventions in limiting the dopaminergic neuronal cell death in these models is discussed with evidence suggesting that the further investigation of their use as part of multi-targeted clinical trials is warranted.

Neurobiology of Aging, 2014

Interleukin-1b Interleukin-6 a b s t r a c t A neuro-inflammatory response has been implicated in... more Interleukin-1b Interleukin-6 a b s t r a c t A neuro-inflammatory response has been implicated in human patients and animal models of Alzheimer's disease (AD). Type-1 interferons are pleiotropic cytokines involved in the initiation and regulation of the pro-inflammatory response; however, their role in AD is unknown. This study investigated the contribution of type-1 IFN signaling in the neuro-inflammatory response to amyloid-beta (Ab) in vitro and in the APP/PS1 transgenic mouse model of AD. Enzyme-linked immunosorbent assay confirmed a 2-fold increase in IFNa in APP/PS1 brains compared with control brains. Quantitative polymerase chain reaction also identified increased IFNa and IFNb expression in human pre-frontal cortex from AD patients. In vitro studies in primary neurons demonstrated Ab-induced type-1 IFN expression preceded that of other classical pro-inflammatory cytokines, IL1-b, and IL-6. Significantly, ablation of type-1 interferon-a receptor 1 expression in BE(2)M17 neuroblastoma cells and primary neurons afforded protection against Ab-induced toxicity. This study supports a role for type-1 interferons in the pro-inflammatory response and neuronal cell death in AD and suggests that blocking type-1 interferon-a receptor 1 maybe a therapeutic target to limit the disease progression.

Microcirculation, 2011

The aim of this study was to assess the ability of Gpx1 to regulate leukocyte-endothelial cell in... more The aim of this study was to assess the ability of Gpx1 to regulate leukocyte-endothelial cell interactions in the cerebral microcirculation under inflammatory conditions associated with oxidative stress. To induce cerebral inflammation, wild-type and Gpx1(-/-) mice underwent systemic treatment with TNF or transient focal cerebral ischemia via MCAO. Leukocyte rolling and adhesion in cerebral postcapillary venules were assessed by intravital microscopy. Absence of Gpx1(-/-) resulted in increased cerebral oxidant production in response to TNF. Under these conditions, leukocyte rolling in cerebral venules was significantly elevated in Gpx1(-/-) mice, whereas leukocyte adhesion was lower than that in wild-type mice. Despite this, expression of key adhesion molecules did not differ between the strains. Following MCAO, Gpx1(-/-) mice displayed significant reductions in rolling and adhesion associated with severe blood flow restriction. In contrast, following treatment with the anti-oxidant ebselen to equalize postischemic cerebral blood flow in wild-type and Gpx1(-/-) mice, absence of Gpx1 was associated with significant elevations in leukocyte interactions. These data show that under some inflammatory conditions, Gpx1 regulates leukocyte-endothelial cell interactions in the cerebral microvasculature, but that this is affected by the nature of the inflammatory insult.

Laboratory Investigation, 2011

The neurovascular unit provides a dynamic interface between the circulation and central nervous s... more The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage.

Journal of Neuroscience Research, 2006

The enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxide... more The enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxides. To test for the consequences of GPx deficiency in astrocytes, astrocyte-rich primary cultures from wild-type and GPx1-deficient [GPx1(-/-)] mice were exposed to H(2)O(2). In GPx1(-/-) astrocytes, the clearance rate of H(2)O(2) was slower than in wild-type cells. In contrast to GPx1-deficient astrocytes, wild-type cells exhibited, within 2 min of H(2)O(2) application, a rapid and transient accumulation of cellular glutathione disulfide that amounted to 60% of total glutathione. The peroxide treatment did not affect the viability of wild-type astrocytes, whereas 45% of the GPx1(-/-) cells died within 8 hr. However, the viability of both types of astrocytes was strongly compromised by lowering cellular glutathione content before peroxide application. In contrast, inactivation of catalase caused substantial cell death only in GPx1(-/-) cells but not in wild-type astrocytes. The cell death observed was prevented by the iron chelators deferoxamine, 1,10-phenathroline, or 2,2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-dipyridyl, whereas preincubation with ferric ammonium citrate increased the toxicity of peroxide treatments. These results demonstrate that GPx1 contributes to the rapid clearance of H(2)O(2) by mouse astrocytes and that both GPx1 and a high concentration of glutathione are required to protect these cells from iron-dependent peroxide damage.

Journal of Neuroinflammation, 2014

Background: Hypoxic-ischaemic injuries such as stroke and traumatic brain injury exhibit features... more Background: Hypoxic-ischaemic injuries such as stroke and traumatic brain injury exhibit features of a distinct neuro-inflammatory response in the hours and days post-injury. Microglial activation, elevated pro-inflammatory cytokines and macrophage infiltration contribute to core tissue damage and contribute to secondary injury within a region termed the penumbra. Type-1 interferons (IFNs) are a super-family of pleiotropic cytokines that regulate pro-inflammatory gene transcription via the classical Jak/Stat pathway; however their role in hypoxia-ischaemia and central nervous system neuro-inflammation remains unknown. Using an in vitro approach, this study investigated the role of type-1 IFN signalling in an inflammatory setting induced by oxygen glucose deprivation (OGD).

Journal of Neurochemistry, 2008

Journal of Neurochemistry, 2005

We have previously identified an increased susceptibility of glutathione peroxidase-1 (Gpx1)-/-mi... more We have previously identified an increased susceptibility of glutathione peroxidase-1 (Gpx1)-/-mice to neuronal apoptosis following mid-cerebral artery (MCA) occlusion. This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. This was addressed in both an in vitro and in vivo model of oxidative stress in the form of exogenous H 2 O 2 and cerebral ischaemia, respectively. Increased levels of cell death were detected in primary neurons lacking Gpx1 following the addition of exogenous H 2 O 2 . This increased apoptosis correlated with a downregulation in the activation of the phospho-inositide 3-kinase [PI(3)K]-Akt survival pathway. The importance of this pathway in protecting against H 2 O 2 -induced cell death was highlighted by the increased susceptibility of wildtype neurons to apoptosis when treated with the PI(3)K inhibitor, LY294002. The Gpx1-/-mice also demonstrated elevated neuronal cell death following MCA occlusion. Although Akt phosphorylation was detected in the Gpx1-/-brains, activation was not seen in later reperfusion events, as demonstrated in wildtype brains. Previous studies have highlighted the importance of Akt phosphorylation in protecting against neuronal cell death following cerebral ischaemia-reperfusion. Our results suggest that the increased susceptibility of Gpx1-/-neurons to H 2 O 2 -induced apoptosis and neuronal cell death in vivo following cerebral ischaemia-reperfusion injury can be attributed in part to diminished activation of Akt. Perturbations in key anti-apoptotic mechanisms as a result of an altered redox state may have implications in the study of oxidative stress-mediated neuropathologies.

Journal of Neurochemistry, 2001

Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxida... more Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxidative state. Recently, unregulated oxidative state has been implicated as detrimental to neural cell viability and involved in both acute and chronic neurodegeneration. In this study we have addressed the importance of a functional glutathione peroxidase in a mouse ischemia/reperfusion model. Two hours of focal cerebral ischemia followed by 24 h of reperfusion was induced via the intraluminal suture method. Infarct volume was increased three-fold in the glutathione peroxidase-1 (Gpx-1) ±/± mouse compared with the wild-type mouse; this was mirrored by an increase in the level of apoptosis found at 24 h in the Gpx-1 ±/± mouse compared with the wild-type mouse.

Journal of Cerebral Blood Flow & Metabolism, 2003

The authors hypothesized that glutathione peroxidase-1 (Gpx-1) contributes to the neuroprotection... more The authors hypothesized that glutathione peroxidase-1 (Gpx-1) contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic (Sod-1 tg) mouse. To investigate this hypothesis, they crossed the Gpx-1 -/-mouse with the Sod-1 tg and subjected the cross to a mouse model of ischemia/reperfusion. Two hours of focal cerebral ischemia followed by 24 hours of reperfusion was induced via intraluminal suture. The Sod-1 tg/Gpx-1 -/-cross exhibited no neuroprotec-tion when infarct volume was measured; indeed, infarct volume increased in the Sod-1 tg/Gpx-1 -/-cross compared with the wild-type mouse. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to ischemia/reperfusion injury. Abbreviations used: Gpx-1, glutathione peroxidase-1; MCA, middle cerebral artery; ROS, reactive oxygen species; Sod-1, superoxide dismutase-1; Sod-1 tg, superoxide dismutase-1 transgenic.

Human Molecular Genetics, 2002

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the e... more Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the expansion of a trinucleotide repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This disorder is characterized by degeneration of motor and sensory neurons, proximal muscular atrophy, and endocrine abnormalities, such as gynecomastia and reduced fertility. We describe the development of a transgenic model of SBMA expressing a full-length human AR (hAR) cDNA carrying 65 (AR 65 ) or 120 CAG repeats (AR 120 ), with widespread expression driven by the cytomegalovirus promoter. Mice carrying the AR 120 transgene displayed behavioral and motor dysfunction, while mice carrying 65 CAG repeats showed a mild phenotype. Progressive muscle weakness and atrophy was observed in AR 120 mice and was associated with the loss of a-motor neurons in the spinal cord. There was no evidence of neurodegeneration in other brain structures. Motor dysfunction was observed in both male and female animals, showing that in SBMA the polyglutamine repeat expansion causes a dominant gain-of-function mutation in the AR. The male mice displayed a progressive reduction in sperm production consistent with testis defects reported in human patients. These mice represent the first model to reproduce the key features of SBMA, making them a useful resource for characterizing disease progression, and for testing therapeutic strategies for both polyglutamine and motor neuron diseases.

Glia, 2006

Organic hydroperoxides are produced in the eicosanoid metabolism and by lipid peroxidation. To ex... more Organic hydroperoxides are produced in the eicosanoid metabolism and by lipid peroxidation. To examine the contribution of glutathione peroxidase-1 (GPx1) and glutathione (GSH) in the disposal of organic hydroperoxides in brain astrocytes, primary astrocyte cultures from wild type or GPx1-deficient (GPx1(-/-)) mice were exposed to cumene hydroperoxide (CHP). After application of 100 microM CHP, the peroxide disappeared quickly from the incubation medium of wild type cells with a half-life of 9 min, whereas CHP clearance was strongly retarded in GPx1(-/-) astrocytes. Depletion of GSH by pre-incubation with buthionine sulfoximine (BSO) significantly slowed CHP clearance by wild type astrocytes, while almost completely preventing peroxide disposal by GPx1(-/-) cells. In contrast, the catalase inhibitor 3-aminotriazole (3AT) had no effect on CHP clearance. Application of CHP to wild type astrocytes was followed by a rapid and transient accumulation of GSSG, whereas in GPx1(-/-) cells no increase in the GSSG content was detected. Astrocytes from both mouse lines remained viable for up to 24 h following CHP exposure, however depletion of cellular GSH by pre-treatment with BSO compromised the viability of astrocytes, an effect that was stronger in GPx1(-/-) than in wild type cells. This cell death was almost completely prevented by iron chelators, whereas pre-incubation with iron increased CHP toxicity. These novel data demonstrate that the toxicity of organic hydroperoxides in astrocytes is iron-mediated, and that an intact GSH system is required for the effective removal of organic hydroperoxides and for protection from these peroxides.